INHIBITION OF MAMMALIAN SQUALENE SYNTHETASE-ACTIVITY BY ZARAGOZIC ACID A IS A RESULT OF COMPETITIVE-INHIBITION FOLLOWED BY MECHANISM-BASED IRREVERSIBLE INACTIVATION

Squalene synthetase (SQS, EC 2.5.1.21) catalyzes the first committed step in the formation of cholesterol and thus represents an ideal site for selectively inhibiting sterol formation. Previous studies have demonstrated that the fungal metabolite, zaragozic acid A (ZGA-A), inhibits SQS activity by mimicking the substrate farnesyl pyrophosphate, the reaction intermediate presqualene pyrophosphate, or both, through a process that confers increased apparent potency in the presence of reduced enzyme concentrations, an observation consist ent with either tight binding reversible competitive inhibition or mechanism-based irreversible inactivation. The studies outlined in this report provide multiple lines of evidence indicating that ZGA-A acts as a mechanism-based irreversible inactivator of SQS. 1) Inhibition of SQS by ZGA-A is dependent on the [SQS] present in the incubation reaction, and this inhibition is time dependent and follows pseudo-first order reaction kinetics, exhibiting hobs values that range between 2 x 10(-4)/s and 23 x 10(-4)/s for [ZGA-A] within the log-linear range of the inhibition curve, and a bimolecular rate constant of 2.3 x 10(5) M(-1) s(-1). 2) SQS activity is titratable by ZGA-A, such that for each [ZGA-A] evaluated, inactivation exhibits a threshold [SQS] whereby enzyme activity at lower [SQS] is totally inhibited. 3) Time dependent inactivation exhibits saturation kinetics with a K-m for the process of 2.5 nM, which is approximately equal to the IC50 for SQS inhibition under these conditions, suggesting that inactivation results from selective modification of a functional group of the enzyme active center rather than from a nonspecific bimolecular reaction mechanism and that most, if not all of the inhibition results from irreversible inactivation. 4) Saturable, time-dependent inactivation occurs with similar inactivation kinetics for both the microsomal and trypsin-solubilized forms of the enzyme, indicating that irreversible inactivation by ZGA-A is not a consequence of membrane modification but is a direct effect of the inhibitor on the enzyme. 5) Inactivation is biphasic, exhibiting a rapid (''burst'') phase followed by a second, pseudo-first order phase, similar to that previously noted for irreversible inactivators in other enzyme systems, and occurs even in the presence of 5 mM concentrations of the nucleophylic scavenger dithiothreitol, suggesting that the reaction between ZGA-A and SQS occurs at or near the active center prior to diffusion of reactive species out of the catalytic cleft. 6) Inactivation can be prevented through competition with the substrate, farnesyl pyrophosphate, further identifying the active center as the site of modification. 7) The inhibitory effects of ZGA-A cannot be reversed either by lowering the [ZGA-A] to noninhibitory levels by dilution or by removal of unreacted ZGA-A from the enzyme by repetitive centrifugation, suggesting that enzyme modification is covalent. 8) The alpha,beta-unsaturated carbonyl moiety of ZGA-A, which is similar to related Michael acceptor moieties present in a variety of mechanism-based inactivators identified in other enzyme systems, appears to be required for eliciting the time dependent irreversible inactivation of SQS by ZGA-A and related alpha,beta-unsaturated carbonyl-containing analogs. 9) Observations similar to those described above were not noted for any of a number of other competitive inhibitors of SQS from a variety of diverse structural classes, whose K-i values for SQS inhibition ranged from 3 to 700 nM, suggesting that the mechanism-based irreversible inactivation of SQS by ZGA-A appears to be specific to this class of compounds rather than to inhibition of SQS per se. These observations strongly suggest that inhibition of SQS by ZGA-A and related analogs occurs through a mechanism-based irreversible inactivation process, presumably with formation of a covalent ZGA-A-SQS adduct that is catalytically incompetent. ZGA-A and related analogs may thus serve as useful tools for probing the substrate binding sites and catalytic reaction centers within the active center of SQS and for further evaluating the unique catalytic mechanisms of this important enzyme.